Mapping the Protein Domain Structures of the Respiratory Mucins: A Mucin Proteome Coverage Study

Abstract

Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides which greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and, to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from ten different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or “spontaneous cleavages”. Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20 which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.

Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure

“…Typically, it is these exposed regions in the N and C termini and the Cys region of MUC5AC and MUC5B against which antibodies have been generated for immunologic quantitation of mucins. A problem is that these regions are also the regions exposed to endogenous (17,33) and exogenous (25) proteolytic activities, which have been reported to cleave these regions of mucin macromolecules (18,25). Thus, these considerations suggest that there may be major technical difficulties measuring mucin concentrations by immunologic techniques in biologic solutions containing active proteases.…”

“…PAS-rich mucin fractions were pooled, reduced, alkylated, and subjected to trypsin digestion. Tryptic peptides were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a Waters Micro-Q mass spectrometer for mucin coverage (33,35,45).…”

Loss of Core 1-derived O-Glycans Decreases Breast Cancer Development in Mice

“…Under non-denaturing and non-reduc- ing condition, immunoblots revealed a single band Ͼ 250 kDa, which is equivalent to the fully-glycosylated Muc1 (22). The molecular weight of Muc1 from ME C1galt1 Ϫ/Ϫ breast tissues was lower relative to WT, suggesting decreased O-glycosylation (Fig.…”

“…However, as the predominant O-glycoprotein, Muc1 is known to play a critical role in mammary gland development in both human and mouse. Muc1 is comprised of two subunits, Muc1-N and Muc1-C (22). The larger subunit (Muc1-N) of the ectodomain primarily consists of tandem repeats, which are heavily O-glycosylated.…”

Proteomic analysis of pediatric sinonasal secretions shows increased MUC5B mucin in CRS

“…The unique number of peptides and spectral counts for MUC5AC and MUC5B, which are similar in molecular weight and therefore comparable (13), are shown in Table 3. MUC5B abundance by spectral count average was significantly higher than MUC5AC spectral count average (P = 0.043) in CRS secretions (Figure 3).…”